Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties

Article abstract of DOI:10.1021/acs.jmedchem.0c01398

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Full text of DOI:10.1021/acs.jmedchem.0c01398

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Article
Discovery of IPN60090, a Clinical Stage Selective Glutaminase‑1
(
GLS-1) Inhibitor with Excellent Pharmacokinetic and
Physicochemical Properties
Michael J. Soth,* Kang Le, Maria Emilia Di Francesco, Matthew M. Hamilton, Gang Liu, Jason P. Burke,
Chris L. Carroll, Jeffrey J. Kovacs, Jennifer P. Bardenhagen, Christopher A. Bristow, Mario Cardozo,
Barbara Czako, Elisa de Stanchina, Ningping Feng, Jill R. Garvey, Jason P. Gay, Mary K. Geck Do,
Jennifer Greer, Michelle Han, Angela Harris, Zachary Herrera, Sha Huang, Virginia Giuliani,
Yongying Jiang, Sarah B. Johnson, Troy A. Johnson, Zhijun Kang, Paul G. Leonard, Zhen Liu,
Timothy McAfoos, Meredith Miller, Pietro Morlacchi, Robert A. Mullinax, Wylie S. Palmer, Jihai Pang,
Norma Rogers, Charles M. Rudin, Hannah E. Shepard, Nakia D. Spencer, Jay Theroff, Qi Wu, Alan Xu,
Ju Anne Yau, Giulio Draetta, Carlo Toniatti, Timothy P. Heffernan, and Philip Jones
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01398
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sı Supporting Information
ABSTRACT: Inhibition of glutaminase-1 (GLS-1) hampers the
proliferation of tumor cells reliant on glutamine. Known
glutaminase inhibitors have potential limitations, and in vivo
exposures are potentially limited due to poor physicochemical
properties. We initiated a GLS-1 inhibitor discovery program
focused on optimizing physicochemical and pharmacokinetic
properties, and have developed a new selective inhibitor,
compound 27 (IPN60090), which is currently in phase 1 clinical
trials. Compound 27 attains high oral exposures in preclinical
species, with strong in vivo target engagement, and should robustly
inhibit glutaminase in humans.
INTRODUCTION
there are multiple reports demonstrating additive or
synergistic effects of GLS-1 inhibition in tumor cells when
combined with a variety of agents in both in vitro and in vivo
■
Glutaminase (GLS-1), an enzyme that converts glutamine to
glutamate, is a promising therapeutic target in tumors
dependent on glutamine.
the production of multiple biochemical intermediates required
for cellular proliferation and maintenance, including gluta-
thione (involved in cellular redox balance), α-ketoglutarate
5
,9−14
1
−4
models.
GLS-1 inhibition has also been reported to
Inhibition of GLS-1 should affect
15,16
sensitize tumor cells to radiation.
There are two known isoforms of glutaminase: GLS-1 (also
called kidney-type or KGA), and GLS-2 (also called liver-type
or LGA). GLS-2 expression appears limited primarily to the
liver, while GLS-1 is ubiquitous. As mentioned above, GLS-1
has been validated as a cancer target, and our program focused
on selective GLS-1 inhibitors.
(
tricarboxylic acid cycle intermediate), and ammonia (build-
ing block for protein synthesis) (Figure 1). In addition, by
increasing the amount of glutamine in the tumor micro-
environment, glutaminase inhibition may help support
activation and proliferation of T-cells to combat tumor
GLS-1 can exist as two variants (KGA and GAC), both of
which appear susceptible to the same classes of inhibitors.
5
,6
growth.
A growing pool of evidence suggests that tumors rewire
metabolic pathways to sustain their needs for energy and
cellular building blocks, and that these changes can increase
reliance on GLS-1. For example, we and others have disclosed
that certain tumors characterized by loss or mutations in
Kelch-like ECH-associated protein (KEAP) and/or NRF2
genes, which encode for proteins that regulate glutathione
Received: August 9, 2020
7
,8
production, are sensitive to GLS-1 inhibition. Additionally,
©
XXXX American Chemical Society
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A
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32 33
30
31
University of Technology, AstraZeneca, Pfizer, Agios,
34
and Rhizen. Calithera’s efforts have progressed the furthest,
with CB-839 (telaglenastat, 4) currently in pivotal clinical
2
6,35
trials for multiple oncology indications.
Compound 4 is a
potent inhibitor of GLS-1, although its physicochemical and
PK properties appear to require fairly high doses (recom-
3
6−38
mended phase 2 doses of 600−800 mg, twice a day).
Both preclinical and clinical results for compound 4 are
encouraging for the use of GLS-1 inhibitors in combination
therapies against multiple cancer types.
We also selected compound 2 as an initial lead and focused
on improving upon its metabolic stability and solubility
profile. Our program prioritized properties in the hope that an
eventual candidate would be able to achieve high sustained
exposures relative to potencies in the clinic, and thus be
positioned to test the clinical potential of maximally inhibiting
glutaminase. We describe herein the efforts that culminated in
the discovery of IPN60090, a compound with excellent
physicochemical and pharmacokinetic properties, currently in
phase 1 clinical trials for solid tumors.
Figure 1. Glutamine metabolism through GLS-1. GLS-1 converts
glutamine to glutamate and ammonia. The produced glutamate can
be further converted by the cellular machinery to glutathione (which
regulates reactive oxygen species (ROS)) or α-ketoglutarate (α-KG)
(
key building block in the tricarboxylic acid (TCA) cycle). The
produced ammonia is a building block for cellular protein syntheses.
There are three main classes of GLS-1 inhibitors reported,
represented by the irreversible and unselective active-site
1
7
inhibitor 6-diazo-5-oxy-L-norleucine (DON, 1) and the
GLS-1-selective allosteric inhibitors bis-2-(5-phenylacetami-
RESULTS AND DISCUSSION
■
1
8
Lead Identification. Our strategy started with consid-
eration of a published crystal structure of compound 2 bound
do-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES, 2)
and
19
compound 968 (3) (Figure 2).
Compound 1, first reported to inhibit glutamine catabolism
by Willis and Seegmiller, inhibits multiple glutamine-
utilizing enzymes including glutaminase, and its polypharma-
cology presumably underlies unacceptable toxicities observed
in the clinic, although efforts are ongoing to potentially
overcome these issues through the use of targeted prodrugs
with low dosing regimens. Compound 3, reported by
Cerione and co-workers, appears to operate through a unique
allosteric mechanism; information on exact binding modes is
currently limited, but the compound has been reported to
39
to full-length human GLS-1 (GAC isoform). Compound 2
binds at the interface of GLS-1 molecules in a 4:2 protein/
inhibitor ratio, with the two aminothiadiazole moeities making
key hydrogen-bonding interactions with the protein. We
therefore embarked on a broad empirical exercise testing
aminothiadiazole alternatives expected to present similar
patterns of hydrogen-bond donors and acceptors. We
1
7
20
4
0
discovered a variety of unique GLS-1 inhibitors; this
manuscript focuses on the series that produced our clinical
candidate, containing an amidotriazole linked to an
acylaminopyridazine. Figure 3 shows our proposed chemical
series and its hypothesized binding mode to the targeted site
of GLS-1. For the sake of discussion, we label the various
portions of the scaffold as “cores”, “linker”, and “wings”.
The acylaminopyridazine core is a known motif in GLS-1
inhibitors and is present in 4; pyridazine can be considered an
isostere of thiadiazole, in the same way that benzene and
21−23
inhibit both GLS-1 and GLS-2.
18
Compound 2, first disclosed in a patent from Elan and
inhibition mechanism elucidated by Robinson and co-
2
4
workers, also has a unique allosteric binding mode, which
25
has been elucidated with protein crystallography. Com-
pound 2 is reported to have high selectivity over GLS-2, and
has served as a chemical starting point for multiple discovery
2
6
27
41
efforts, including those of Johns Hopkins University,
thiophene are considered to be isosteres of each other.
8
28,29
Calithera Biosciences, University of Pittsburgh,
Zhejiang
Amidotriazole cores are, to our knowledge, unprecedented in
Figure 2. Structures of known GLS inhibitors DON (1), BPTES (2), “compound 968” (3), and CB-839 (4).
B
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39
Figure 3. Proposed binding of series example 5 in the GLS-1 tetramer, modeled using PDB 3UO9.
GLS-1 inhibitors. A comparison of amidotriazole and
acylaminopyridazine fragments suggests a close overlay of
most hydrogen-bond donors and acceptors (Figure 4).
and >58 mL/min/kg in human); second, solubilities were very
low (<1 μM). The low solubilities, and possibly the low
microsomal stabilities, are likely attributable to the large
number of aromatic rings in the compounds; therefore, our
first optimization efforts focused on replacing one of the
aromatic wings with smaller and/or less lipophilic groups.
Based on the liabilities just described, the key data that we
monitored at this early stage were potencies, microsomal
stabilities, and solubilities.
Triazole Wing Exploration. Our scan of triazole
wingpieces focused on replacing the heteroaryl motif of
parent compound 7 (Table 2). All replacements investigated
showed a loss in potency. However, the simple methylamide 8
still retained a reasonable amount of potency, with a higher
Figure 4. Hydrogen-bonding motifs of amidotriazoles overlay well
onto those of aminopyridazines.
43
ligand binding efficiency (0.30 compared to 0.27 for 7).
The four-carbon linker was first popularized by Thangavelu
More importantly, compound 8 showed much improved
microsomal stabilities relative to the original compounds and
literature comparators 2 and 4, especially human. We
therefore favored the methylamide to keep constant as we
probed other substituents on the scaffold. While the
microsomal properties of compound 8 were promising,
solubility remained low.
Pyridazine Wing Exploration. Keeping the triazole wing
constant as a methylamide, we explored pyridazine wings
(Table 3). In the hope of improving solubility, we first
replaced the trifluoromethylbenzyl group of compound 8 with
simple pyridylmethyl side chains (compounds 14 and 15),
which did improve solubilities while retaining most of the
potency of 8. The ortho-pyridine 14 was chosen for further
optimization because of its superior cell potency.
The 4-trifluoromethyl variant (compound 16) was well
tolerated, with only a slight loss of cellular potency compared
to the trifluoromethoxybenzyl analogue 8, and more
importantly retained the improved solubility. We therefore
embarked on a more extensive scan of 4-substituted-2-
pyridylmethyl wingpieces, resulting in many analogues with
good to excellent potencies, low microsomal clearances, and
reasonable solubilities. Particularly noteworthy were analogues
16 and 19, which showed promising overall profiles, and
analogue 20, which while less soluble was especially potent in
our cellular assays.
and co-workers as a substitute for the thioether linker present
4
2
in 2, and was suggested by modeling and early empirical
studies to be an optimal length for our series. We were able to
further improve upon this linker, as will be described later in
the manuscript. The wings are tolerant of a wide variety of
modifications and were a major focus of our optimization
efforts.
Scaffold Proof of Concept. For proof of potency for this
novel scaffold, we synthesized compounds 5, 6, and 7, which
contain benzyl, or pyridylmethyl and trifluoromethoxybenzyl
wings, similar to those present in compounds 2 and 4.
Enzyme potencies were evaluated in a GLS-1 (GAC isoform)
enzyme assay indirectly measuring the conversion of
glutamine to glutamate, and cellular potencies were evaluated
in a cellular viability assay (A549 cell line). For comparison
purposes, we include data from the same assays for
compounds 2 and 4. All three of our new compounds
demonstrate good potencies in enzyme and cell-based assays
Table 1), thus giving us confidence in exploring the scaffold
further.
Further profiling of compounds 5−7 revealed two major
liabilities, also issues for literature comparators 2 and 4, which
were the primary foci of our lead optimization efforts (Table
(
1
). First, microsomal stabilities were low (most Cl ’s ranked
int
as high clearance by internal criteria, >83 mL/min/kg in rat
C
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Table 1. Scaffold Proof of Concept
a
Inhibition of purified recombinant human GAC assessed via a dual-coupled enzyme assay. IC50 values are reported as the mean of at least three
b
determinations, except for compound 5 (n = 1). Standard deviations and counts are included in the Supporting Information. Inhibition of
proliferation of A549 cells. IC50 values are reported as the mean of at least three determinations, except for compound 5 (n = 2). Standard
c
d
deviations and counts are included in the Supporting Information. Liver microsomal intrinsic clearance (mL/min/kg protein). Kinetic aqueous
solubility (μM) in phosphate buffer (100 mM) at pH 7.4; shaken for 1 h at RT.
Linker: Benefits of Fluorine. We were interested in
probing the effects of fluorine substitution on potencies and
microsomal stabilities. Molecular modeling suggested that a
while compound 25 is significantly more potent than
compound 27, we believe its higher in vivo clearances and
low oral exposures could present challenges at later stages of
development, and therefore favored the less potent compound
27. Compound 27 has moderate solubility in pH 7 buffer and
simulated intestinal fluid, but high solubility in simulated
gastrointestinal fluid. These solubility results were attained for
the crystalline freebase form that was carried into develop-
ment. The compound shows mild efflux in a Caco-2 assay,
used to assess intestinal permeability; this mild efflux potential
has a minimal effect on oral absorption, based on the high
bioavailabilities observed with oral dosing.
44
fluorine substituent would be well tolerated on the linker,
45
specifically on either of the two internal carbons. For
synthetic reasons, we focused on fluorination of the internal
carbon closer to the triazole core. Fluorination is not only
tolerated, but consistently improved both cell potencies (albeit
moderately) and microsomal stabilities (Table 4). Fluorina-
tion introduces a chiral center; we focused on the R-
enantiomer because it often showed small potency advantages
(
e.g., compound 21 vs compound 22).
We also found that introduction of an ortho-methyl group
Deeper Profiling of Compound 27 (IPN60090). A
summary of potency, selectivity, and pharmacokinetic proper-
ties of compound 27 is shown in Figure 5. Compound 27 is a
highly selective inhibitor of GLS-1, with no activity observed
against GLS-2. Testing in a cellular target engagement assay,
which measured the conversion of glutamine to glutamate,
showed a similar IC50 to that seen in the cellular viability
assay. As summarized in Figure 5, compound 27 showed no
significant inhibition of the hERG channel or common
cytochrome P450 (CYP) enzymes. Compound 27 also
showed no significant activities against an 80-member
Eurofins CEREP panel of various ion channels and receptors,
or against a 97-member Eurofins DiscoverX kinase panel.
Specific results from the CEREP and DiscoverX panels are
contained in the Supporting Information.
to the pyridine could, in select cases, slightly improve
potencies (compound 26 vs compound 23). While the
ortho-methyl group of compound 27 did not improve
potencies relative to the des-methyl analogue 24, ultimately
7 showed superior in vivo properties.
Final Triaging. Based primarily on physicochemical and
metabolic properties coupled with cellular potencies, we
selected compounds 24, 26, and 27 for further character-
ization. In addition, we profiled analogue 25, on the rationale
that this example, despite overall poorer properties, may have
a lower bar on required exposures due to its excellent
potencies. We triaged the set based on solubilities,
permeabilities, microsomal stabilities, and in vivo PK (rat
and dog). Tables 5 and 6 show summaries of in vitro and in
vivo PK (rat and dog) data for these compounds.
2
The pharmacokinetic properties of compound 27 in rat and
dog are superior to its properties in mouse and monkey, with
rat and dog showing lower in vivo clearances and 5−6 times
longer half-lives. Because of the low volumes of distribution
Compound 27 was chosen for further evaluation based on
its overall profile, including significantly longer half-lives in rat
and dog relative to the other leading compounds. Importantly,
D
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Table 2. Structure−Activity Relationship (SAR) on the Triazole Wing: Benzyl Replacements
a
Inhibition of purified recombinant human GLS-1 (GAC isoform) assessed via a dual-coupled enzyme assay. IC50 values are reported as the mean
b
of at least three determinations. Standard deviations and counts are included in the Supporting Information. Inhibition of proliferation of A549
cells. IC50 values are reported as the mean of at least three determinations. Standard deviations and counts are included in the Supporting
c
d
Information. Liver microsomal intrinsic clearance (mL/min/kg protein). Kinetic aqueous solubility (μM) in phosphate buffer (100 mM) at pH
.4; shaken for 1 h at RT.
7
across species, half-lives are short unless clearances are
extremely low, as is the case for rat and dog. Clearances
across species are qualitatively in agreement with in vitro
metabolic stabilities. Given that the in vitro clearances in
human microsomes and hepatocytes are very low (similar to
or lower than values for rat and dog), we expect low in vivo
clearances and favorable half-lives in humans, more similar to
rat and dog than to mouse and monkey.
Compound 27 was further tested in ascending single-dose
oral PK experiments in mouse, rat, and dog (Figure 6). Across
the dose ranges tested (up to 200 mg/kg in mouse, 100 mg/
kg in rat, and 10 mg/kg in dog), maximal concentrations and
total exposures continually increased with dose, with high
exposures achieved across species.
In Vivo Target Engagement of Compound 27. To
demonstrate that 27 could robustly inhibit glutaminolysis in
vivo, and to determine plasma concentrations required for in
vivo target engagement, the compound was orally adminis-
tered in an H460 non-small cell lung cancer xenograft model
at doses of 10, 50, or 250 mg/kg (Figure 7). To determine
target engagement, we analyzed tumor samples at the 8 and
concentrations observed at these doses and time points were
0.058−0.316 μM (Figure 7b).
In Vivo Efficacy of Compound 27. In a benchmarking
study detailed in the Supporting Information (Figures S1 and
S2), we compared compound 27 to clinical stage compound 4
(
Calithera’s CB-839) in an H2122 non-small cell lung cancer
cell line-derived xenograft mouse model. The results from that
study indicated that compound 27 dosed orally at 100 mg/kg
twice daily shows similar efficacy and target engagement to
46
compound 4 dosed orally at 250 mg/kg twice daily.
To demonstrate antitumor efficacy in an additional model,
which was also used to confirm a combination therapy
hypothesis, 27 was tested in an Ru337 non-small cell lung
cancer patient-derived xenograft (PDX) mouse model (Figure
8
). Compound 27 demonstrates robust in vivo target
engagement when orally dosed to this model at doses of
5−100 mg/kg twice daily, at both 4 and 28 days (see
2
Supporting Information Figure S3). We tested the compound
at oral doses of 100 mg/kg twice daily, as both a monotherapy
and in combination with the dual TORC1/2 inhibitor TAK-
47
2
28 (also known as MLN0128, INK128). This combination
2
4 h time points for changes in glutamine and glutamate
was chosen based on results from translational biology studies
concentrations relative to vehicle control, on the expectation
that inhibition of glutaminase should increase levels of
glutamine relative to glutamate. Figure 7a shows the ratio of
glutamate to glutamine in tumors when the animals were
treated with ascending doses of compound 27. The expected
changes were indeed observed, with the effect at 50 mg/kg
similar to that at 250 mg/kg despite an increase in plasma
concentrations, suggesting that we are maximally inhibiting
target at doses of 50−250 mg/kg. The average free plasma
to define rational combination strategies (manuscript under
48
revision).
As a single agent, 27 caused a 28% tumor growth inhibition.
This result is consistent with other reported preclinical studies
9
,10,13,31
on GLS inhibitors as monotherapies.
The effects of
TAK-228 administration were likewise modest at the dose
tested (41% tumor growth inhibition). However, the
combination regimen caused an 85% tumor growth inhibition,
E
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Table 3. SAR of Pyridazine Wing: Pyridylmethyls
a
Inhibition of purified recombinant human GLS-1 (GAC isoform) assessed via a dual-coupled enzyme assay. IC50 values are reported as the mean
b
of at least three determinations. Standard deviations and counts are included in the Supporting Information. Inhibition of proliferation of A549
cells. IC50 values are reported as the mean of at least three determinations. Standard deviations and counts are included in the Supporting
c
d
Information. Liver microsomal intrinsic clearance (mL/min/kg protein). Kinetic aqueous solubility (μM) in phosphate buffer (100 mM) at pH
7.4; shaken for 1 h at RT.
an improvement over either single agent. The results strongly
support testing this combination clinically.
methylamide 31, which was subsequently converted to varied
final products 14−20.
Chemistry. The general synthetic routes for the prepara-
tion of compounds are shown in Schemes 1−5. Scheme 1
shows an early synthesis, which was particularly helpful for
exploring variations in the triazole wing. 4-Azido-1-propyne
was reacted with tert-butyl propiolate in a selective azide-
alkyne Huisgen cyclization to afford triazole 28, and the
remaining alkyne was then coupled with 1-iodo-4-amino-
pyridazine to afford alkynyl aminopyridazine 29. Alkyne 29
was reduced to alkyl aminopyridazine 30, which was
converted to varied amide intermediates 31. Finally, the tert-
butyl esters of intermediates 31 were deprotected to the
corresponding acids, which were coupled with amines to
afford final products 5−13.
Synthesis of fluorinated analogues relied on intermediate
iodide 32, the synthesis of which is shown in Scheme 3. 2-
((Benzyloxy)methyl)oxirane was reacted with sodium azide to
afford azido alcohol 33, which was reacted with tert-butyl
propiolate in an azide-alkyne Huisgen cyclization to afford
triazole alcohol 34. Alcohol 34 was converted to fluoride 35,
and the benzyl ether of 35 was removed to afford new alcohol
36. The alcohol of intermediate 36 was converted via
intermediate tosylate 37 to the corresponding iodide 32.
Most of the compounds in this manuscript were synthesized
as R-enantiomers, starting from (R)-2-((benzyloxy)methyl)-
oxirane as shown in the scheme, with the exception of 22,
which followed the same synthesis except starting from (S)-2-
((benzyloxy)methyl)oxirane.
Scheme 2 shows the synthesis used to explore variations in
the pyridazine wing, in which the triazole wing was kept
constant as a methylamide. The tert-butyl ester of alkyl
aminopyridazine 30 was deprotected to the corresponding
acid, which was coupled with methylamine to afford
Scheme 4 shows one synthetic route to our fluorinated
analogues. 3,6-Dichloropyridazine was first treated with
hydroiodic acid, and the resulting 3,6-diiodopyridazine was
reacted with the anion of tert-butyl malonate to selectively
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Table 4. Linker Fluorination and Pyridine Methylation
a
Inhibition of purified recombinant human GLS-1 (GAC isoform) assessed via a dual-coupled enzyme assay. IC50 values are reported as the mean
b
of at least three determinations. Standard deviations and counts are included in the Supporting Information. Inhibition of proliferation of A549
cells. IC50 values are reported as the mean of at least three determinations. Standard deviations and counts are included in the Supporting
c
d
Information. Liver microsomal intrinsic clearance (mL/min/kg protein). Kinetic aqueous solubility (μM) in phosphate buffer (100 mM) at pH
7
e
.4; shaken for 1 h at RT. Thermodynamic aqueous solubility (μM) in phosphate buffer (100 mM) at pH 7.0; shaken for 1 h then equilibrated
overnight at RT.
afford mono-substituted malonate 38. Malonate 38 was then
coupled with intermediate iodide 32 to afford di-substituted
malonate 39. Di-substituted malonate 39 was then treated
with trifluoroacetic acid to remove all three tert-butyl esters,
with in situ bis-decarboxylation of the alkyl linker, and the
remaining carboxylic acid was coupled with methylamine to
afford pyridazine iodide 40. The pyridazine iodide was then
coupled with varied primary amides, under palladium catalysis
conditions, to afford final products 21−23, 25 or 26.
The final coupling step shown in Scheme 4 was sometimes
problematic due to the generally poor solubility of
intermediate 40. The alternative route shown in Scheme 5
circumvented this issue by instead performing the iodopyr-
idazine-amide coupling on tri-ester 39 (to afford intermediate
alkyl linker, and the product acid 42 was then coupled with
methylamine to afford final compounds 24 and 27.
CONCLUSIONS
■
Based on its overall profile, compound 27 (IPN60090) was
selected for development and taken into IND-enabling
studies. Additional translational research was conducted to
inform the future clinical program and is being reported
43
elsewhere. Importantly, while this candidate was not the
most potent analogue created in the series, its excellent
physicochemical and pharmacokinetic properties outweighed
potency considerations. Compound 27 is currently in the
clinic, and robust target engagement is being achieved in
human subjects.
41), which was treated with hydrogen chloride to remove all
three tert-butyl esters with in situ bis-decarboxylation of the
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Table 5. In Vitro Properties of Select Compounds
a
Thermodynamic aqueous solubility (μM) in phosphate buffer (100 mM) at pH 7.0, fed-state simulated intestinal fluid (FeSSIF) at pH 5.0, or
b
fasted-state simulated gastrointestinal fluid (SGF) at pH 1.6; shaken for 1 h then equilibrated overnight at RT. Thermodynamic aqueous
solubilities for the crystalline most stable polymorph of 27, identified during later development, in phosphate buffer (50 mM) at pH 6.8, FeSSIF or
c
SGF, shaken for 24 h at 25 °C (phosphate buffer) or 37 °C (FeSSIF and SGF). Permeability in Caco-2 cells AB (apical to basolateral) and BA
−
6
d
(
(
basolateral to apical) movement of 10 μM test compound in 21-day cultured Caco-2 cells ((cm/s) × 10 ). Liver microsomal intrinsic clearance
e f
mL/min/kg protein). Liver hepatocyte intrinsic clearance (mL/min/kg protein). Plasma protein binding (PPB) determined by equilibrium
dialysis.
system with a Waters Xbridge Prep C18 5 μm OBD, 19 mm × 150
mm or 50 mm × 100 mm column and an SQ detector mass
spectrometer with electrospray ionization (ESI) ionization. The
identity and purity of inhibitors were confirmed by NMR
spectroscopy and ultraperformance liquid chromatography (UPLC)
coupled to low-resolution mass spectrometry (LRMS) and were
≥95%. High-resolution mass spectrometry (HRMS) data were
collected for leading compounds 24−27. NMR spectra were
recorded on Bruker Instruments operating at 500 or 600 MHz.
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commer-
cial sources and were used without further purification. Chromato-
graphic purification of compounds was performed by column
chromatography utilizing a Biotage system applying Biotage SNAP
columns with Biotage KP-Sil silica or Biotage Zip Si columns with
Biotage KP-Sil silica, or a Teledyne ISCO system with RediSep Rf
normal phase silica cartridges. Other compounds were purified by
preparative HPLC in reverse-phase mode using a Waters Autopurify
H
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Table 6. In Vivo Properties of Select Compounds
a
Cmax, area under the curve (AUC) , and F% were determined from the oral dose, and CL, Vd , and t were determined from the IV dose.
last
ss
1/2
b
c
d
Dosed as a hydrochloride salt. Dosed as a bis-hydrochloride salt. Male Sprague Dawley (SD) rats. Doses were 0.3 mg/kg IV (intravenous) and 3
e
mpk PO (per os), except for compound 25 (0.3 mpk IV, 2 mpk PO). Male beagle dogs. Doses were 0.3 mg/kg IV (intravenous) and 3 mpk PO
per os).
(
NMR spectra were obtained as CDCl , CD OD, D O, (CD ) SO,
the mixture was added the above-prepared 3,6-diiodopyridazine
3
3
2
3 2
(
CD ) CO, C D , or CD CN solutions (reported in ppm), using
(75.00 g, 226.0 mmol), and the reaction mixture was stirred at reflux
3
2
6
6
3
tetramethylsilane (0.00 ppm) or residual solvent (CDCl , 7.26 ppm;
for 8 h. The reaction mixture was quenched with a sat. aq NH Cl
3
4
CD OD, 3.31 ppm; D O, 4.79 ppm; (CD ) SO, 2.50 ppm;
solution (500 mL) and extracted with 1:1 EtOAc/petroleum ether (3
3
2
3 2
(
CD ) CO, 2.05 ppm; C D , 7.16 ppm; CD CN, 1.94 ppm) as the
× 500 mL). The combined organic layers were dried over Na SO ,
3
2
6
6
3
2
4
reference standard. Low-resolution mass spectra were obtained on
either a Waters H-class UPLC with a Waters Acquity UPLC BEH
C18 1.7 μm, 2.1 mm × 50 mm column, UV detection between 200
and 400 nm, evaporating light scattering detection, and an SQ
detector mass spectrometer with ESI ionization; or a Water I class
UPLC with a Waters Acquity UPLC CSH C18 1.7 μm, 2.1 mm × 50
mm column, UV detection at 254 and 290 nm, evaporating light
scattering detection, and an SQ detector 2 mass spectrometer with
ESI ionization. High-resolution mass spectra on leading compounds
filtered, and concentrated under reduced pressure. The residue was
purified by SiO gel chromatography (10:1 petroleum ether/EtOAc)
2
to give di-tert-butyl 2-(6-iodopyridazin-3-yl)malonate (83.00 g, 87%)
+
as a white solid. MS (ES ) C H IN O requires: 420, found: 421
1
5
21
2
4
+
1
[M + H] . H NMR (DMSO-d ) δ 8.20 (d, J = 8.8 Hz, 1H), 7.47 (d,
J = 8.8 H, 1H), 5.03 (s, 1H), 1.43 (s, 18H). C NMR (DMSO-d ) δ
6
1
3
6
165.86, 156.63, 137.92, 129.60, 127.16, 83.03, 59.95, 27.90.
(S)-tert-Butyl 1-(2-fluoro-3-iodopropyl)-1H-1,2,3-triazole-4-
carboxylate (32). Step 1: (R)-1-Azido-3-benzyloxy(propan-2-ol)
(33). To a solution of (R)-2-((benzyloxy)methyl)oxirane (2.423 mL,
2
4−27 were obtained on a Waters Acquity I-Class UPLC−Thermo
LTQ Orbitrap Elite MS instrument, with a Waters Acquity UPLC
BEH C18 1.7 μm, 2.1 mm × 100 mm column, ESI ionization.
Di-tert-butyl 2-(6-iodopyridazin-3-yl)malonate (38). Step 1:
15.89 mmol) and NH Cl (1.70 g, 31.8 mmol) in MeOH (39.5 mL)
4
and water (5.92 mL) was added sodium azide (5.17 g, 79.0 mmol),
and the resulting mixture was stirred at RT overnight. The mixture
was concentrated under reduced pressure, and the residue was
partitioned between EtOAc (50 mL) and water (60 mL). The two
layers were separated, and the aqueous layer was extracted with
EtOAc (3 × 50 mL). The organic layers were combined, dried over
3
4
4
,6-Diiodopyridazine. A mixture of 3,6-dichloropyridazine (60.00 g,
02.7 mmol) and 55% aqueous hydrogen iodide solution (30.30 mL,
02.7 mmol) was stirred at 90 °C for 12 h. A solid was isolated by
filtration and then suspended in a sat. aq NaHCO solution (300
3
mL). The solid was isolated by filtration and washed with petroleum
ether (2 × 200 mL) to give crude 3,6-diiodopyridazine (120.0 g,
9
MgSO , filtered, and concentrated under reduced pressure to give
4
(R)-1-azido-3-(benzyloxy)propan-2-ol (3.01 g, 91%) as a colorless
+
+
0%) as a yellow solid, which was used without further purification.
oil. MS (ES ) C H N O requires: 207, found: 208 [M + H] .
10 13 3 2
+
+
MS (ES ) C H I N requires: 332, found: 333 [M + H] .
Step 2: (R)-tert-Butyl 1-(3-Benzyloxy)-2-hydroxypropyl-1H-1,2,3-
triazole-4-carboxylate (34). To a solution of the above-prepared
(R)-1-azido-3-(benzyloxy)propan-2-ol (3.01 g, 14.5 mmol), tert-butyl
propiolate (2.393 mL, 17.43 mmol), DIEA (0.253 mL, 1.45 mmol),
and AcOH (0.083 mL, 1.45 mmol) in DCM (58.1 mL) was added
4
2 2
2
Step 2: Di-tert-butyl 2-(6-iodopyridazin-3-yl)malonate (38). To
a suspension of NaH (60% in mineral oil, 27.12 g, 678.0 mmol) in
THF (750 mL) was added di-tert-butyl propanedioate (100.8 mL,
4
52.0 mmol), and the mixture was stirred at 28 °C for 15 min. To
I
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a
Figure 5. Summary of potencies, selectivities, and pharmacokinetic properties of compound 27. All IC values are reported as the mean of at
5
0
b
least two determinations. Inhibition of purified recombinant human GLS-1 (GAC isoform) assessed via a dual-coupled enzyme assay. IC value
5
0
c
is the mean ± standard deviation for 36 determinations. Inhibition of purified recombinant human GLS-2 assessed via a dual-coupled enzyme
d
e
assay. Inhibition of proliferation of A549 cells. IC value is the mean ± standard deviation for 36 determinations. Inhibition of cellular
5
0
glutaminase assessed via direct measurement of glutamine and glutamate levels in the cellular media. IC value is the mean ± standard deviation
5
0
f
g
for four determinations. hERG QPatch assay, CHO cell line. Inhibition of metabolism of known substrates for the indicated cytochrome P450
h
i
enzymes. Eurofins CEREP profiling service; details are given in the Supporting Information. Eurofins DiscoverX KINOMEscan profiling service,
scanEDGE product; details are in the Supporting Information. Liver microsomal intrinsic clearance (mL/min/kg protein). Liver hepatocyte
intrinsic clearance (mL/min/kg protein). Plasma protein binding determined by equilibrium dialysis. Dosed as a bis-hydrochloride salt to
female CD-1 mice, male Sprague Dawley rats, male cynomolgus monkeys, and male beagle dogs. Doses were 0.3 mg/kg IV (intravenous) and 3
mpk PO (per os), except for mouse (3 mpk IV, 10 mpk PO). Cmax, AUClast, and F% were determined from the oral dose, and CL, Vd , and t
j
k
l
m
ss
1/2
were determined from the IV dose.
Figure 6. Ascending dose oral PK of compound 27 (bis-hydrochloride salt) in mouse, rat, and dog dosed in 0.5% aqueous methylcellulose
vehicle. Values are the means with standard deviation for three animals except for mouse AUC values, which are composite determinations, as
described in the Experimental Section.
CuI (0.138 g, 0.726 mmol), and the resulting mixture was stirred at
the resulting suspension was filtered and washed with DCM (20 mL)
and EtOAc (20 mL). The filtrate was concentrated under reduced
RT overnight. SiO gel (10 g) was added to the stirring mixture, and
2
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mL) under an atmosphere of N . The solution was purged with N
2
2
for 10 min and then with N still flowing, Pd(OH) on carbon (0.746
2
2
g, 1.06 mmol) was added. The resulting suspension was stirred as it
was purged with H for 2 min. The reaction mixture was then stirred
2
under an atmosphere of H at 1 atm for 12 h, then purged with N ,
2
2
filtered through Celite, and concentrated under reduced pressure to
give crude (S)-tert-butyl 1-(2-fluoro-3-hydroxypropyl)-1H-1,2,3-tri-
azole-4-carboxylate (1.32 g, 101%) as a pale yellow solid, which was
+
used without further purification. (1.32 g, 101%). MS (ES )
+
C H FN O requires: 245, found: 246 [M + H] .
1
0
16
3
3
Step 5: (S)-tert-Butyl 1-(2-Fluoro-3-(tosyloxy)propyl)-1H-1,2,3-
triazole-4-carboxylate (37). To a solution of the above-prepared
S)-tert-butyl 1-(2-fluoro-3-hydroxypropyl)-1H-1,2,3-triazole-4-car-
(
boxylate (1.32 g, 5.38 mmol) and DMAP (0.986 g, 8.07 mmol) in
DCM (26.9 mL) was added 4-methylbenzene-1-sulfonyl chloride
(
1.23 g, 6.46 mmol), while the solution was maintained at RT by a
water bath. The resulting mixture was stirred at RT for 1.5 h, then
diluted with EtOAc (100 mL), and washed with sat. aq NH Cl (2 ×
4
4
0 mL). The organic layer was dried over MgSO , filtered, and
4
concentrated under reduced pressure to give crude (S)-tert-butyl 1-
(
2-fluoro-3-(tosyloxy)propyl)-1H-1,2,3-triazole-4-carboxylate (1.803
+
g, 84%), which was used without further purification. MS (ES )
C H FN O S requires: 399, found: 400 [M + H] .
+
1
7
22
3
5
Figure 7. In vivo target engagement and concentrations of
compound 27 in an H460 NSG mouse xenograft model. Compound
Step 6: (S)-tert-Butyl 1-(2-Fluoro-3-iodopropyl)-1H-1,2,3-tria-
zole-4-carboxylate (32). To a solution of the above-prepared (S)-
tert-butyl 1-(2-fluoro-3-(tosyloxy)propyl)-1H-1,2,3-triazole-4-carbox-
ylate (2.12 g, 5.31 mmol) in acetone (26.5 mL) was added sodium
iodide (0.796 g, 5.31 mmol), and the resulting mixture was stirred at
27 (bis-hydrochloride) was administered orally at the indicated doses
in a 0.5% aqueous methylcellulose vehicle. There were six animals
per group. (a) Glutamate/glutamine ratios at 8 and 24 h after each
dose, with standard deviation bars; (b) free plasma concentrations,
with standard deviations, of compound 27 at 8 and 24 h for each
dose.
8
0 °C for 3 h. Additional sodium iodide (1.6 g) was added, and the
mixture was stirred at 90 °C for 2 h. The mixture was allowed to cool
to RT, then diluted with 1:1 EtOAc/hexanes (150 mL), and
sequentially washed with water (2 × 50 mL) and a sat. aq. NaCl
solution (50 mL). The organic layer was dried over MgSO , filtered,
pressure to give crude (R)-tert-butyl 1-(3-(benzyloxy)-2-hydrox-
ypropyl)-1H-1,2,3-triazole-4-carboxylate (3.95 g, 82%) as an orange
4
and concentrated under reduced pressure. The residue was adsorbed
onto Celite and purified by SiO2 gel chromatography (0−50%
EtOAc/hexanes) to give (S)-tert-butyl 1-(2-fluoro-3-iodopropyl)-1H-
+
oil, which was used without further purification. MS (ES )
+
C H N O requires: 333, found: 334 [M + H] .
17
23
3
4
1
,2,3-triazole-4-carboxylate (1.71 g, 91%) as a white crystalline solid.
Step 3: (S)-tert-Butyl 1-(3-Benzyloxy)-2-fluoropropyl-1H-1,2,3-
+
+ 1
triazole-4-carboxylate (35). To a 0 °C solution of the above-
prepared (R)-tert-butyl 1-(3-(benzyloxy)-2-hydroxypropyl)-1H-1,2,3-
triazole-4-carboxylate (3.95 g, 11.8 mmol) and pyridine (1.909 mL,
MS (ES ) C10
H15FIN O
3
2
requires: 355, found: 356 [M + H] . H
NMR (DMSO-d
) δ 8.69 (s, 1H), 5.09−4.95 (m, 1H), 4.87−4.72
6
1
3
(m, 2H), 3.62−3.54 (m, 1H), 3.48−3.40 (m, 1H), 1.54 (s, 9H).
C
23.70 mmol) in DCM (23.70 mL) was added DAST (3.13 mL, 23.7
NMR (DMSO-d ) δ 159.87. 140.57, 130.12, 90.42 (d, J = 178.3 Hz),
6
mmol). The resulting mixture was stirred at RT for 2.5 h, then
81.80, 53.52 (d, J = 21.4 Hz), 28.32, 4.26 (d, J = 21.5 Hz).
2-(4-(3,3-Difluorocyclobutoxy)-6-methylpyridin-2-yl)-
acetamide. Step 1: 2-Chloro-4-(3,3-difluorocyclobutoxy)-6-meth-
ylpyridine. To a suspension of 3,3-difluorocyclobutanol (590 mg,
filtered through a plug of SiO gel, and rinsed with DCM (50 mL).
2
The filtrate was concentrated under reduced pressure, and the
residue was adsorbed onto Celite and purified by SiO chromatog-
2
5
.46 mmol) and 2-chloro-6-methyl-4-nitropyridine (0.660 mL, 5.38
mmol) in THF (5.5 mL) was added cesium carbonate (3562.4 mg,
0.93 mmol), and the resulting orange-yellow mixture was stirred at
raphy (0−50% EtOAc in hexanes) to give (S)-tert-butyl 1-(3-
(
4
3
benzyloxy)-2-fluoropropyl)-1H-1,2,3-triazole-4-carboxylate (1.781 g,
+
1
5%) as a tan crystalline solid. MS (ES ) C H FN O requires:
17 22 3 3
+
35, found: 336 [M + H] .
65 °C for 24 h. The resulting brown mixture was allowed to cool,
then diluted with 11 mL of EtOAc, and filtered through Celite 545,
rinsing well with ethyl acetate. The dark yellow filtrate was
concentrated to give crude 2-chloro-4-(3,3-difluorocyclobutoxy)-6-
methylpyridine (1231.5 mg, 98%) as a dark yellow oil, which was
Step 4: (S)-tert-Butyl 1-(2-Fluoro-3-hydroxypropyl)-1H-1,2,3-
triazole-4-carboxylate (36). A reaction vessel was charged with
the above-prepared (S)-tert-butyl 1-(3-(benzyloxy)-2-fluoropropyl)-
1H-1,2,3-triazole-4-carboxylate (1.78 g, 5.31 mmol) and EtOAc (53.1
Figure 8. In vivo efficacies in an Ru337 PDX subcutaneous mouse tumor model of compound 27 as monotherapy and in combination with dual
TORC inhibitor TAK-228. Compound 27 (neutral compound) was administered orally twice daily at 100 mg/kg in an 0.5% aqueous
methylcellulose vehicle; TAK-228 was dosed orally once daily at 1 mg/kg in an 0.5% aqueous methylcellulose, 5% sucrose vehicle. Both
49
compounds were dosed on a schedule of 5 days on/2 days off, and there were eight animals per group. (a) Tumor volumes plotted as mean ±
standard deviation; (b) average % bodyweights relative to day 0 plotted as mean ± standard deviation.
K
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a
Scheme 1. General Synthesis of Compounds 5−13
a
Reagents and conditions: (i) copper sulfate, ascorbic acid, tert-butanol/water (54%); (ii) bis(triphenylphosphine) palladium(II) dichloride,
copper iodide, triethylamine, tetrahydrofuran (THF), 60 °C (84%); (iii) Raney nickel, hydrogen, methanol (89%); (iv) R-CO H,
2
propanephosphonic acid anhydride, pyridine (25−78%); (v) (a) trifluoroacetic acid (TFA), dichloromethane (DCM), (b) R-NH , amide
2
coupling reagent, solvent (15−98%).
a
Scheme 2. General Synthesis of Compounds 14−20
a
Reagents and conditions: (i) (a) trifluoroacetic acid, dichloromethane, (b) methylamine, N,N-diisopropylethylamine (DIEA), 1-
[
(
bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, tetrahydrofuran/N,N-dimethylformamide
29%); (ii) R-CO H, amide coupling reagent, solvent (5−82%).
2
+
used without further purification. MS (ES ) C H ClF NO requires:
layers were dried over Na SO , filtered, and concentrated to give 2-
2 4
1
0
10
2
+
233, found: 234 [M + H] .
(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamide (133.0
mg, 87%) as an off-white solid, which was used without further
Step 2: tert-Butyl 2-Cyano-2-(4-(3,3-difluorocyclobutoxy)-6-
+
methylpyridin-2-yl)acetate. A mixture of the above-prepared 2-
chloro-4-(3,3-difluorocyclobutoxy)-6-methylpyridine (487 mg, 2.08
mmol), tert-butyl 2-cyanoacetate (0.456 mL, 3.13 mmol), cesium
carbonate (2048.6 mg, 6.29 mmol), and chloro(2-di-tert-butylphos-
phino-2′.4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl]-
palladium(II) (74.1 mg, 0.108 mmol) in toluene (20 mL) was
purification. MS (ES ) C H F N O requires: 256, found: 257 [M
1
2
14
2
2
2
+
1
+ H] . H NMR (DMSO-d ) δ 7.46 (br. s, 1H), 6.94 (br. s, 1H),
6
6
.69−6.68 (m, 2H), 4.84−4.78 (m, 1H), 3.46 (s, 2H), 3.27−3.19
13
(m, 2H), 2.74−2.65 (m, 2H), 2.38 (s, 3H). C NMR (DMSO-d ) δ
6
1
1
71.57, 163.79, 159.43, 157.74, 119.43 (dd, J = 281.5, 270.9 Hz),
08.55, 107.88, 61.69 (dd, J = 17.0, 10.3 Hz), 45.13, 42.89 (appar. t,
degassed by blowing N through a needle for 5 min, then put under
N in an air condenser, heated to 110 °C over 5 min, and stirred for
h. The orange mixture was allowed to cool, then concentrated to a
yellow-orange solid. The residue was purified via SiO2 gel
chromatography (0−100% EtOAc in hexanes) to give tert-butyl 2-
cyano-2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetate
2
J = 22.8 Hz), 24.52.
2
(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-
2-yl)acetamido) Pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-
1,2,3-triazole-4-carboxamide (27). Step 1: (R)-di-tert-Butyl 2-
(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-
1
2
-(6-iodopyridazin-3-yl)malonate (39). A mixture of potassium
carbonate (0.412 g, 2.98 mmol), di-tert-butyl 2-(6-iodopyridazin-3-
yl)malonate (38) (1.25 g, 2.98 mmol), and (S)-tert-butyl 1-(2-fluoro-
3-iodopropyl)-1H-1,2,3-triazole-4-carboxylate (32) (1.00 g, 2.82
mmol) in a vial was degassed and then treated with DMF (9.39
(
555.6 mg, 79%) as a yellow solid.
Step 3: 2-(4-(3,3-Difluorocyclobutoxy)-6-methylpyridin-2-yl)-
acetamide. A flask with stirbar was sequentially charged with
water (0.296 mL), conc. aq HCl (5.616 mL), and tert-butyl 2-cyano-
2
-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetate (203.0
mL). The mixture was degassed and backfilled with N for three
2
mg, 0.600 mmol). The yellow mixture was stirred at RT for 4 h,
in which time it became a yellow solution. The solution was chilled
in an ice/water bath, then treated with 30 mL of a 2.0 M aqueous
NaOH solution and further adjusted to pH 7 using 2.0 M aqueous
NaOH (3−4 mL) then 1 M aqueous HCl (0.5−1 mL). The solution
was extracted with EtOAc (3 × 15 mL), and the combined organic
cycles and then stirred at 25 °C for 80 h. The mixture was diluted
with EtOAc/hexanes (1:1, 200 mL) and washed with water (2 × 100
mL). The combined aqueous layers were extracted with EtOAc/
hexanes (1:1, 100 mL). The combined organic layers were
concentrated, washed with a sat. aq NaCl solution, and concentrated
under reduced pressure. The residue was purified by SiO2 gel
L
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a
Scheme 3. Synthesis of Intermediate Iodide 32
a
Reagents and conditions: (i) sodium azide, ammonium chloride, methanol/water (91%); (ii) tert-butyl propiolate, N,N-diisopropylethylamine,
acetic acid, dichloromethane (82% crude); (iii) (diethylamino)sulfur trifluoride (DAST), pyridine, dichloromethane (45%); (iv) 10% palladium
hydroxide on carbon, hydrogen, ethyl acetate (101% crude); (v) 4-toluenesulfonyl chloride, 4-dimethylaminopyridine (DMAP), dichloromethane
(84% crude); sodium iodide, acetone (91%).
,
a b
Scheme 4. General Synthesis of Compounds 21−23, 25, and 26
a
Reagents and conditions: (i) (a) 55% aqueous hydrogen iodide, 90 °C (90%), (b) di-tert-butyl propanedioate, sodium hydride, tetrahydrofuran,
6
5 °C (78%); (ii) intermediate 32, potassium carbonate, N,N-dimethylformamide (75%); (iii) (a) trifluoroacetic acid, dichloromethane, 40 °C, (b)
methylamine, N,N-diisopropylethylamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate,
b
tetrahydrofuran/N,N-dimethylformamide (56%); (iv) R-CONH , palladium-catalyzed coupling, solvent (15−57%). Compound 22, the
enantiomer of compound 21, was made using the same synthetic route including preparation of starting material shown in Scheme 3, but starting
from (S)-2-((benzyloxy)methyl)oxirane in place of (R)-2-((benzyloxy)methyl)oxirane.
2
chromatography (5−60% EtOAc in hexanes) to give (R)-di-tert-butyl
chromatography (0−3% MeOH in DCM) to give di-tert-butyl (R)-2-
(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-
(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)-
pyridazin-3-yl)malonate (36.5 g, 86%) as a foamy yellow solid. MS
2
2
-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-
-(6-iodopyridazin-3-yl)malonate (1.36 g, 75%) as a yellow liquid.
+
+
MS (ES ) C H FIN O requires: 647, found: 648 [M + H] .
25
35
5
6
+
+
Step 2: Di-tert-butyl (R)-2-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-
triazol-1-yl)-2-fluoropropyl)-2-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-
methylpyridin-2-yl)acetamido)pyridazin-3-yl)malonate (41). A
degassed solution of (R)-di-tert-butyl 2-(3-(4-(tert-butoxycarbonyl)-
(ES ) C H F N O requires: 775, found: 776 [M + H] .
37 48 3 7 8
Step 3: (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyr-
idin-2-yl)acetamido) Pyridazin-3-yl)-2-fluorobutyl)-1H-1,2,3-tria-
zole-4-carboxylic Acid (42). A solution of (R)-di-tert-butyl 2-(3-(4-
(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-(2-
(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)-
pyridazin-3-yl)malonate (36.0 g, 46.4 mmol) in 4.0 M HCl in 1,4-
dioxane (696.0 mL, 2784 mmol) was stirred at 70 °C for 16 h. A
white precipitate formed. The reaction mixture was allowed to cool
to RT, and the precipitate was isolated by filtration, washed with
EtOAc, and dried in vacuo to give crude (R)-1-(4-(6-(2-(4-(3,3-
difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido) pyridazin-3-
1
H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-iodopyridazin-3-yl)-
malonate (39) (42.4 g, 65.6 mmol), 2-(4-(3,3-difluorocyclobutoxy)-
-methylpyridin-2-yl)acetamide (14.0 g, 54.6 mmol), cesium
6
carbonate (35.6 g, 109 mmol), Xantphos (6.32 g, 10.9 mmol), and
allylpalladium chloride dimer (1.00 g, 2.73 mmol) in 1,4-dioxane
(300 mL) was stirred at 70 °C for 16 h. The reaction mixture was
allowed to cool to RT, then filtered, and the filtrate was concentrated
under reduced pressure. The residue was purified by SiO2 gel
M
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a
Scheme 5. General Synthesis of Compounds 24, 27
a
Reagents and conditions: (i) R-CONH palladium-catalyzed coupling, solvent (34−86%); (ii) hydrogen chloride, dioxane; (iii) methylamine,
2
,
N,N-diisopropylethylamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, tetrahydrofuran/
N,N-dimethylformamide (two-step 56−68%).
yl)-2-fluorobutyl)-1H-1,2,3-triazole-4-carboxylic acid as an off-white
solid, which was used without further purification in the next step.
(60 mL) at 0 °C was added HCl in dioxane (4.0 M, 11.93 mL, 47.70
mmol), and the resulting mixture was stirred for 5 min and then
concentrated under reduced pressure. The residue was redissolved in
acetonitrile (ACN) and water, lyophilized, and the resulting solid was
triturated with EtOAc and dried in vacuo to give (R)-1-(4-(6-(2-(4-
+
+
MS (ES ) C H F N O requires: 519, found: 520 [M + H] .
23
24
3
7
4
Step 4: (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyr-
idin-2-yl)acetamido) Pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-
1
,2,3-triazole-4-carboxamide (27). To a solution of crude (R)-1-
(
3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido) pyrida-
(
4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)-
zin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide
bis-hydrochloride (14.03 g, 97%) as a white solid. MS (ES )
C H F N O requires: 532, found: 533 [M + H] . H NMR
+
acetamido)pyridazin-3-yl)-2-fluorobutyl)-1H-1,2,3-triazole-4-carbox-
ylic acid hydrochloride prepared in the previous step (assumed 46.4
mmol) in DMF (200 mL) at 0 °C were added 1-
+
1
2
4
27
3
8
3
(
8
DMSO-d ) δ 11.66 (s, 1 H), 8.53 (s, 1 H), 8.47 (q, J = 5.3, 1 H),
.23 (d, J = 9.1 Hz, 1 H), 7.73 (d, J = 9.5 Hz, 1 H), 7.44 (d, J = 2.0
6
[
3
(
5
bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]- pyridinium
-oxide hexafluorophosphate (HATU; 17.64 g, 46.4 mmol), DIEA
40.5 mL, 232 mmol), and 2.0 M methylamine in THF (27.8 mL,
5.7 mmol). The resulting mixture was stirred at 20 °C for 1 h and
Hz, 1 H), 7.39 (d, J = 2.0 Hz, 1 H), 4.96−5.11 (m, 2 H), 4.67−4.86
(m, 2 H), 4.36 (s, 2 H), 3.34 (m, 2 H), 3.07 (m, 2 H), 2.87 (m, 2
H), 2.76 (d, J = 4.9 Hz, 3 H), 2.68 (s, 3 H), 2.24−1.95 (m, 2 H).
The compound (2 mg/mL, 10 μL per injection) was analyzed on a
Shimadzu Prominence HPLC system with a Lux Cellulose 4 column
(4.6 mm × 150 mm, 5 μM, 1 mL/min) using a mobile phase of
water/acetonitrile (50:50) and showed an enantiomeric excess of
>98%. Retention time: 11.3 min.
then concentrated under reduced pressure. Water (1000 mL) and
DCM (500 mL) were added, and the layers were separated. The
aqueous layer was extracted with DCM (3 × 300 mL), and the
combined organic layers were washed with sat. aq NaCl, dried over
MgSO , filtered, and concentrated under reduced pressure. The
4
residue was purified by SiO gel chromatography (0−8% MeOH in
In Vitro Pharmacology. GLS-1 Enzyme Assay. The glutaminase
dual-coupled fluorescence assay was performed in a 384-well, black,
Greiner, nonbinding plate (Greiner, Catalog #784900) with assay
buffer consisting of 50 mM Hepes (pH 7.4) (Gibco, Catalog #15630-
080), 250 μM ethylenediaminetetraacetic acid (EDTA; pH 8)
(Calbiochem, Catalog #4055), and 0.12 mM Triton-X 100 (Sigma,
Catalog #T9284). All final concentrations refer to a 20 μL volume.
Stock solutions of the test compounds were prepared in 100%
dimethyl sulfoxide (DMSO; Sigma, Catalog #D2650) and serially
diluted 1:3 using 100% DMSO. Compounds were additionally
diluted 1:50 in assay buffer, and 5 μL/well were transferred to the
assay plate. A 4× stock of glutaminase and potassium phosphate
dibasic trihydrate (Sigma, Catalog #P5504) diluted in assay buffer
was preincubated at RT for 10 min. Glutaminase with potassium
phosphate dibasic trihydrate (5 μL/well) was added to the
microplate (final concentrations 2 nM and 50 mM, respectively)
followed by a 10 min incubation at RT. The coupled reaction
consisted of glutamate oxidase (Sigma, Catalog #G5921), Amplex
UltraRED (Molecular Probes, Catalog #A36006), glutamine (Sigma,
Catalog #G3126), and horseradish peroxidase (Sigma, Catalog
#P8375). Glutamate oxidase (5 μL/well) and Amplex UltraRED
diluted in assay buffer (final concentrations 100 mU/mL and 75 μM,
respectively) and 5 μL/well of glutamine and horseradish peroxidase
diluted in assay buffer (final concentrations 1 mM and 100 mU/mL,
respectively) were added to the microplate under subdued light
2
DCM) to give (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-meth-
ylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-
1
H-1,2,3-triazole-4-carboxamide (16.8 g, 68%) as a white solid.
+
+
HRMS (ES ) C H F N O requires: 533.2231 [M + H] , found:
2
4
28
3
8
3
+
1
5
1
9
5
33.2229 [M + H] . H NMR (DMSO-d ) δ 11.30 (s, 1 H), 8.51 (s,
6
H), 8.47 (q, J = 4.4, 1 H), 8.22 (d, J = 9.1 Hz, 1 H), 7.60 (d, J =
.3 Hz, 1 H), 6.79 (d, J = 2.5 Hz, 1 H), 6.72 (d, J = 2.5 Hz, 1 H),
.09−4.96 (m, 1 H), 4.90−4.70 (m, 3 H), 3.87 (s, 2 H), 3.28−3.18
(
(
m, 2 H), 3.08−2.98 (m, 2 H), 2.76 (d, J = 4.9 Hz, 3 H), 2.75−2.63
13
m, 2 H), 2.39 (s, 3H), 2.20−1.95 (m, 2 H). C NMR (DMSO-d )
6
δ 169.58, 163.45, 160.01, 159.21, 158.70, 156.32, 154.13, 142.99,
1
9
28.42, 126.88, 118.94 (dd, J = 281.7, 270.4 Hz), 108.52, 107.73,
1.25 (d, J = 172.3 Hz), 62.31 (dd, J = 16.98, 10.69 Hz), 52.99 (d, J
=
21.4 Hz), 45.14, 42.41 (appar. t, J = 22.6 Hz), 31.04 (d, J = 20.1
Hz), 30.07 (d, J = 3.8 Hz), 25.53, 24.07. An assumed bis-
hydrochloride salt was used in in vivo experiments described in the
text, as well as to confirm enantiopurity using a chiral HPLC method
validated with mixtures of the title compound and its (S) enantiomer
(synthesized in the same way but starting with (S)-2-((benzyloxy)-
methyl)oxirane in place of (R)-2-((benzyloxy)methyl)oxirane). The
bis-hydrochloride salt was made as follows: To a solution of (R)-1-
(
4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)-
acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-
-carboxamide (12.71 g, 23.87 mmol) in MeOH (20 mL) and DCM
4
N
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Journal of Medicinal Chemistry
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Article
followed by a 20 min incubation at RT. The resorufin signal was
measured using a PerkinElmer Envision plate reader: excitation, 535
nm; emission, 590 nm. IC50 values were calculated using a four-
parameter logistic curve fit using Genedata Screener software.
GLS-2 Enzyme Assay. The GLS2 dual-coupled fluorescence assay
was performed in a 384-well, black, Greiner, nonbinding plate
diluted 1:200 in culture medium, and 200 μL/well were transferred
to the tissue culture plate. Following compound addition, the
microplate was incubated at 37 °C for 24 h. The L-glutamine and L-
glutamate levels in the media were then measured using the YSI 2900
Biochemistry Analyzer (Xylem). IC50 values were calculated using a
four-parameter logistic curve fit using Genedata Screener software.
In Vitro Metabolic Stability, Solubility, Caco-2 Perme-
ability, Plasma Protein Binding, hERG and CYP Inhibition
Experiments. Microsomal Stability. Microsomal stability assays
were conducted on a Beckmann Biomek FXp laboratory automation
system. The liver microsomal incubation mixture consisted of liver
microsomes (0.5 mg microsomal protein/mL), the test compound (1
(
Greiner, Catalog #784900) with assay buffer consisting of 50 mM
Hepes (pH 7.4) (Gibco, Catalog #15630-080), 250 μM EDTA (pH
) (Calbiochem, Catalog #4055), and 0.12 mM Triton-X 100
Sigma, Catalog #T9284). All final concentrations refer to a 20 μL
volume. Stock solutions of the test compounds were prepared in
8
(
100% DMSO (Sigma, Catalog #D2650) and serially diluted 1:3 using
100% DMSO. Compounds were additionally diluted 1:50 in assay
μM), MgCl (3 mM), and EDTA (1 mM) in potassium phosphate
2
buffer (100 mM, pH 7.4). Midazolam and Ketanserin were used as
the assay control substrates. The reaction was initiated with the
addition of a nicotinamide adenine dinucleotide phosphate
buffer, and 5 μL/well were transferred to the assay plate. A 4× stock
of GLS-2 and potassium phosphate dibasic trihydrate (Sigma,
Catalog #P5504) diluted in assay buffer was preincubated at RT
for 10 min. GLS-2 with potassium phosphate dibasic trihydrate (5
μL/well) was added to the microplate (final concentrations 33 nM
and 50 mM, respectively) followed by a 10 min incubation at RT.
The coupled reaction consisted of glutamate oxidase (Sigma, Catalog
(
NADPH) regeneration solution (1.3 mM NADPH) and maintained
at 37 °C with shaking. At five time points ranging from 0 to 45 min,
aliquots (50 μL) were removed and quenched with acetonitrile (100
μL) containing an internal standard (imipramine). After vortex and
centrifugation, the samples were analyzed by liquid chromatography
with tandem mass spectrometry (LC-MS/MS). Calculation of the in
#
G5921), Amplex UltraRED (Molecular Probes, Catalog #A36006),
glutamine (Sigma, Catalog #G3126), and horseradish peroxidase
Sigma, Catalog #P8375). Glutamate oxidase (5 μL/well) and
50
(
vitro half-lives and clearance followed literature guidelines.
Amplex UltraRED diluted in assay buffer (final concentrations 100
mU/mL and 75 μM, respectively) and 5 μL/well of glutamine and
horseradish peroxidase diluted in assay buffer (final concentrations 3
mM and 100 mU/mL, respectively) were added to the microplate
under subdued light. The resorufin signal was measured continuously
for 20 min using a PerkinElmer Envision plate reader: excitation, 535
nm; emission, 590 nm. IC50 values were calculated using a four-
parameter logistic curve fit using Genedata Screener software.
A549 Cell Viability Assay. A549 cells were routinely maintained in
filtered Roswell Park Memorial Institute (RPMI) media (Gibco,
Catalog #11875) supplemented with 10% dialyzed fetal bovine serum
Hepatocytes Stability. Hepatocytes were purchased from
Bioreclamation IVT, Xenotech, or RILD. Stock solutions were
prepared at 10 mM in DMSO for the test compound. Aliquots of the
stock solutions were diluted to 200 μM with DMSO and then further
diluted to 2 μM with Krebs−Henseleit buffer (KHB) buffer. The
procedure was as follows: Count hepatocytes and then dilute the cell
6
suspensions to the appropriate density (viable cell density = 2 × 10
cells/mL). Add 50 μL of prewarmed (37 °C) 2 μM test compound
to the wells designated for different time points. For 0 min, add 100
μL of ACN containing internal standard (IS) to the wells followed by
50 μL of hepatocytes solution and then seal the wells. Add 50 μL of
prewarmed hepatocytes solution to the wells designated for 15, 30,
60, and 120 min, and start timing. Place the assay plate in an
incubator at 37 °C. At 15, 30, 60, and 120 min, add 100 μL of ACN
to the wells, respectively, then seal the corresponding wells. After
quenching, sonicate the plate for 5 min and then centrifuge at 5594g
for 15 min (Thermo Multifuge × 3R). Transfer 50 μL of the
supernatant from each well into a 96-well sample plate containing
120 μL of ultrapure water for LC/MS analysis. The peak area
response ratio (PARR) to IS of the compounds at 15, 30, 60, and
120 min was compared to the PARR at 0 min to determine the
percent of the test compound remaining at each time point. Half-
lives were calculated using Excel software, fitting to a single-phase
exponential decay equation.
(
FBS; Corning, Catalog #35-071-CV) using a humidified incubator
37 °C, 5% CO , and ambient O ). In preparation for the viability
(
2
2
assay, the cells were harvested and resuspended in filtered RPMI
media supplemented with 10% dialyzed FBS (Corning, Catalog #35-
0
71-CV). The cells were seeded onto a 384-well, black PerkinElmer
tissue culture plate (PerkinElmer, Catalog #6007660) at a density of
000 cells/well in a volume of 40 μL. The tissue culture plate was
incubated for 24 h at 37 °C with 5% CO and ambient O . Stock
1
2
2
solutions of the test compounds were prepared in 100% DMSO
Sigma, Catalog #D2650) and serially diluted 1:3 using 100%
(
DMSO. Compounds were additionally diluted 1:40 in culture
medium, and 10 μL/well were transferred to the tissue culture
plate. Following compound addition, the microplate was incubated at
3
7 °C for 72 h. Promega’s CellTiter-Fluor reagent (10 μL, Promega,
Kinetic Solubility. Studies were conducted in 100 mM phosphate
buffer (pH 7.4). A stock solution was prepared at 10 mM in DMSO
for each test compound. Stock solutions (10 μL) were added into
990 μL of 100 mM phosphate buffer (pH 7.4) for final
concentrations of test compounds of 100 μM. The assay plate was
shaken on a vibrator (IKA, MTS 2/4) for 1 h at 1000 rpm at RT and
then centrifuged at 12 000 rpm (Thermo Fisher) for 10 min to
precipitate undissolved particles. An aliquot of the supernatant was
taken from each well and diluted 10× with buffer, then analyzed by
LC-MS/MS compared to a standard curve.
Catalog #G6082), GF-AFC substrate diluted in assay buffer, was
added to the plate for a 1× final concentration. 0.5% DMSO and 20
μM etoposide were used as controls to define 100 and 0% viability,
respectively. The plate was then shaken on an orbital shaker at 300
revolutions per minute (RPM) for 15 min at RT followed by a 30
min incubation at 37 °C. The CellTiter-Fluor signal was measured
using a PerkinElmer Envision plate reader: excitation, 400 nm;
emission, 505 nm. IC50 values were calculated using a four-parameter
logistic curve fit using Genedata Screener software.
A549 Target Engagement Assay. A549 cells were routinely
maintained in filtered RPMI media (Gibco, Catalog 11875)
supplemented with 10% dialyzed FBS (Corning, Catalog #35-071-
Thermodynamic Solubility. Studies were conducted in buffers as
listed in Tables 4 and 5. Approximately 2 mg of each test compound
was weighed into a 2 mL vial, and to the vial was then added 1 mL of
buffer. Each sample was vortexed for 30 s to achieve a homogenous
dispersion, and then shaken on a Labquaker for 24 h at RT. If all
solid dissolved, then an additional test compound was added. After
24 h, the visual appearance of each sample was noted, and the sample
was manually filtered through a 0.45 μm nylon filter. The subsequent
filtrate, diluted as necessary with a suitable solvent, was analyzed by
LC-MS/MS compared to a standard curve.
CV) using a humidified incubator (37 °C, 5% CO , and ambient
2
O ). In preparation for the target engagement assay, the cells were
2
harvested and resuspended in filtered RPMI media supplemented
with 10% dialyzed FBS (Corning, Catalog #35-071-CV). The cells
were seeded onto a 96-well tissue culture plate (Falcon, Catalog
#
353072) at a density of 15 000 cells/well in a volume of 100 μL.
The tissue culture plate was incubated for 24 h at 37 °C with 5%
CO and ambient O . Stock solutions of the test compounds were
Caco-2 Permeability. Caco-2 cells were obtained from American
Tissue Culture Collection (Rockville, MD). The cells were
maintained in modified Eagle’s medium (MEM) containing 10%
2
2
prepared in 100% DMSO (Sigma, Catalog #D2650) and serially
diluted 1:3 using 100% DMSO. Compounds were additionally
O
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heat-inactivated FBS and 1% non-essential amino acids, in CO at 37
CYP Inhibition Assays. Studies were carried out in human liver
microsomes (0.1 mg/mL for CYP 1A2/2C9/2D6/3A4; 0.5 mg/mL
for CYP 2C19). Liver microsomes were purchased from BD Gentest.
Aliquots of the compound stock solutions were diluted to 4 mM with
acetonitrile, and then further diluted upon the addition of liver
microsomes (0.2 mg/mL). An aliquot of 30 μL of diluted test
compound solution was mixed with 15 μL of substrate solution. The
plate was prewarmed to 37 °C, then 15 μL of 8 mM NADPH (also
prewarmed to 37 °C) was added. The plate was incubated at 37 °C
for the following incubation times: 5 min for 3A4, 10 min for 1A2/
2C9/2D6, and 45 min for 2C19. The reaction was stopped by adding
acetonitrile at the designated time point. The assay plates were
shaken on a vibrator (IKA, MTS 2/4) for 10 min (600 rpm) and
centrifuged at 5594g for 15 min (Thermo Multifuge × 3R). Aliquots
of the supernatant were taken, diluted 1:3 into distilled water, and
metabolite concentrations were analyzed by LC-MS/MS compared
to internal standards. Substrates used were Phenacetin (30 μM) for
1A2, diclofenac (10 μM) for 2C9, S-Mephenytoin (35 μM) for
2C19, Midazolam (10 μM) or Testosterone (80 μM) for 3A4, and
Bufuralol (10 μM) for 2D6. Metabolites measured were Acetamino-
phen for 1A2, 4′-Hydroxy-Diclofenac for 2C9, Hydroxy-Mepheny-
toin for 2C19, 1-Hydroxy-Midazolam or Osalmid for 3A4, and 1-
Hydroxy-Bufuralol for 2D6. Positive control inhibitors used were α-
Naphthoflavone for CYP1A2, Sulfaphenazole for CYP2C9, Omepra-
zole for CYP2C19, Quinidine for CYP2D6, and Ketoconazole for
CYP3A4. The peak area response ratio (PARR) of metabolite to
internal standard of the metabolites in test compound samples at
desired time points was compared to the PARR in control samples to
determine the percent of control sample (% control) at each time
point. Percent inhibition was calculated as 100% control.
In Vivo Pharmacokinetics. Mouse. Female mice (CD1 strain,
purchased from Shanghai JH Laboratory Animal Co., Ltd.), weighing
20−30 g were used for studies. Food and water were available to all
of the animals ad libitum. Test article was dosed via tail vein (IV
doses) or oral gavage (PO doses), respectively. Blood samples were
collected from all of the animals at predose and at 0.083, 0.25, 0.5, 1,
2, 4, 8, and 24 h postdose into tubes containing the anticoagulant
K2EDTA (three animals per time point with three time points
collected per animal). Plasma was separated from the blood by
centrifugation at 4 °C and stored at −70 °C until analysis. Test
article concentrations in plasma were quantified using a liquid
chromatography with tandem mass spectrometry (LC-MS/MS)
method.
2
°
C. Cells were seeded on polycarbonate filter inserts (Millipore,
CAT#PSHT 010 R5). The cells were cultured for 21−28 days prior
to the transport experiments. The transepithelial electric resistance
(
TEER) and Lucifer Yellow permeability were checked before and
after the assay. Compounds were dissolved at 10 mM in DMSO and
diluted for studies in Hank’s Balanced Salt Solution (HBSS,
Invitrogen, Cat #14025-092) with 25 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), pH 7.4. Compounds were
tested at 10 μM, in both the apical-to-basolateral (A−B) and
basolateral-to-apical (B−A) directions, and were tested at 37 °C for
9
0 min. At the end of incubation, donor samples were diluted 10-fold
by assay buffer, then 60 μL of receiver and diluted-donor samples
were mixed with 60 μL of acetonitrile and concentrations were
analyzed by LC-MS/MS compared to a standard curve.
Plasma Protein Binding. Plasma protein binding (PPB) assays
were conducted using the Rapid Equilibrium Dialysis (RED) device
(Thermo Fisher Scientific). Warfarin and metoprolol were used as
the control substrates. To the receiver side was added 350 μL of
phosphate buffered saline (pH 7.4, 1×, Gibco). To the donor side
was added 200 μL of plasma (Bioreclamation IVT) spiked with the
test compound (5 μM). The same plasma/test compound solution
(50 μL) was also used for the recovery sample. The plate was
covered with Immunoware sealing tape and was incubated at 37 °C
with shaking at 100 rpm for 5 h. After the incubation, both the
receiver and donor sides were sampled (50 μL) and matched with
the same volume of matrix from the other side. The recovery, donor,
and receiver samples were extracted with 300 μL of cold ACN
containing imipramine as the internal standard. After vortex and
centrifugation, the supernatant (150 μL) was subjected to LC-MS
quantitation. PPB (% bound) was calculated as % bound = 100 ×
(
[donor] − [receiver])/[donor].
hERG Qpatch (Automated Electrophysiology) Assay. A CHO
cell line stably transfected with hERG cDNA and expressing hERG
channels was used for studies. The cells were cultured in medium
(from Invitrogen) containing Ham’s F12, 10% (v/v) heat-inactivated
FBS, 100 μg/mL hygromycin B, and 100 μg/mL Geneticin. The cells
used in the QPatch study met the following criteria: under
microscopy examination, the majority of cells in suspension were
single and isolated; their viability was greater than 95%, with only a
few debris and cell clumps (which may clog the holes in QPlate
during whole-cell clamp recording); cell density ranged within 3−8 ×
6
1
0 cells/mL in the final suspension before applying to the QPatch
stir chamber. After leaving the CO2 incubator, the cells were
maintained in serum-free medium buffered with HEPES. The cells
were used for recording within 4 h after harvesting. Whole-cell
recordings were performed using automated QPatch (Sophion,
Denmark). The cells were voltage clamped at a holding potential of
Rat. Male rats (SD strain, purchased from Shanghai JH Laboratory
Animal Co., Ltd.) weighing 200−300 g were used for studies. The
animals were fasted overnight and fed 4 h postdose. Water was
available ad libitum for all of the animals. Test article was dosed via
dorsal foot vein (IV doses) or oral gavage (PO doses). Blood
samples were collected via tail vein from all of the animals at predose
and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes
containing the anticoagulant K2EDTA. Plasma was separated from
the blood by centrifugation at 4 °C and stored at −70 °C until
analysis. Test article concentrations in plasma were quantified using a
liquid chromatography with tandem mass spectrometry (LC-MS/
MS) method.
Dog. Male Beagle dogs (purchased from Beijing Marshall
Biotechnology Co., Ltd.) weighing 7−10 kg were used for studies.
The animals were fasted overnight and fed 4 h postdose. Test article
was administered to dogs via the cephalic vein (IV doses) or oral
gavage (PO doses). Blood samples were collected via the saphenous
vein or cephalic vein from all of the animals at predose and 0.083,
0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes containing the
anticoagulant K2EDTA. Plasma was separated from the blood by
centrifugation at 4 °C and stored at −70 °C until analysis. Test
article concentrations in plasma were quantified using a liquid
chromatography with tandem mass spectrometry (LC-MS/MS)
method.
−80 mV. The hERG current was activated by depolarizing at +20
mV for 5 s, after which the current was taken back to −50 mV for 5 s
to remove the inactivation and observe the outward tail current. The
maximum amount of tail current size was used to determine hERG
current amplitude. After achieving break-in (whole-cell) configu-
ration, the cells were recorded for 120 s to assess current stability.
The voltage protocol described above was then applied to the cells
every 15 s throughout the whole procedure. Only stable cells with
recording parameters above threshold were allowed to enter the drug
application procedure. All experiments were conducted at RT (about
25 °C). External solution containing 0.1% DMSO (vehicle) was
applied to the cells to establish the baseline. After allowing the
current to stabilize for 3 min, the compound was applied. Compound
solution was added, and the cells were kept in the test solution until
the compound’s effect reached a steady state or for a maximum of 4
min. For the dose−response assay, the compound was applied to the
cells accumulatively from low to high concentrations. Washout with
external solution was performed after compound testing. Positive
control cisapride was used to test the same batch of hERG cells for
compound testing to ensure the normal response and quality of cells.
Data were analyzed using Assay Software provided by Sophion,
XLFit, and GraphPad Prism.
Monkey. Male Cynomolgus monkeys (purchased from Hainan
Jingang Biotech. Co., Ltd.) weighing 3−5 kg were used for studies.
The animals were fasted overnight and fed 4 h postdose. Test article
P
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Article
was administered to monkeys via the cephalic vein (IV doses) or
nasal gavage (PO doses). Blood samples were collected via the
saphenous vein or cephalic vein from all of the animals at predose
and 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes
containing the anticoagulant K2EDTA. Plasma was separated from
the blood by centrifugation at 4 °C and stored at −70 °C until
analysis. Test article concentrations in plasma were quantified using a
liquid chromatography with tandem mass spectrometry (LC-MS/
MS) method.
combination arms, standard deviations were very low (bars smaller
than the size of plotted datapoints).
Molecular Modeling. The molecular model in Figure 3 of
compound 5 bound to GLS1 was generated using Molecular
Operating Environment (MOE, 2019.01; Chemical Computing
Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal,
QC, Canada, H3A 2R7, 2019). The X-ray crystal structure of GLS1
with 2 (BPTES) bound (PDB 3UO9) was prepared for modeling
using the QuickPrep routine in MOE. Compound 2 was then
removed from the structure, and 5 was docked using the general
docking module within MOE. The top-scoring ligand pose for 5 that
replicated the binding mode and interactions between BPTES and
GLS1 was subjected to additional energy minimization in MOE,
while holding the protein coordinates fixed to arrive at the final
model.
In Vivo Models. All experiments were conducted in compliance
with institutional guidelines.
Glutamine and Glutamate Levels in H460 Xenograft Model.
NSG female mice (Jackson Labs) were implanted with H460 cells (5
5
×
10 cells/mouse diluted with matrigel 1:1). The mice were
between 6 and 10 weeks old. All of the animals received LabDiet
5
053 chow ad libitum. Tumors were allowed to grow to 300−400
3
mm , and the animals were treated with compound 27 (bis-
ASSOCIATED CONTENT
sı Supporting Information
■
hydrochloride) formulated in 0.5% methylcellulose in sterile water.
*
The animals were euthanized via CO at 8 or 24 h after a single dose
2
of compound 27 by oral gavage, and tumors were harvested. Each
group contained six animals. The tumors were weighed and snap-
frozen. Tumor sections were homogenized using an OmniBEAD
Ruptor 24 at 100 mg tissue/mL in MeOH/Water (80:20) containing
C5-L-glutamine and C5-L-glutamate as internal standards.
Homogenates were centrifuged at 15 000 rpm at 4 °C for 10 min.
An aliquot of 10 μL of the supernatant was diluted with 190 μL of
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01398.
Molecular formula strings (CSV)
1
3
13
Experimental details for the preparation of compounds
5
−26; UPLC and high-resolution mass spectrometry
data for componds 24−27; logD, logP, and pK
a
0.1% formic acid in ACN/water (50:50), vortexed for 15 s, and
determinations for compound 27; experimental proce-
dures for oral dosing formulations and additional in vivo
studies; standard deviations for GLS-1 and A549 assay
results for compounds 2, 4, and 5−27; representative
centrifuged at 15 000 rpm at 4 °C for 5 min. The samples were
analyzed on an Agilent 1290 infinity LC system coupled with an
Agilent 6460 triple quadrupole mass spectrometer operated at
+
positive mode (ESI ). A Waters XBridge Amide column (3.5 μm; 4.6
IC curve for compound 27 in the A549 assay; and
×
100 mm) was used for analyte separation. The HPLC buffer A was
5% (v/v) water/ACN containing 20 mM ammonium hydroxide and
50
9
2
Kinomescan and CEREP off-target screening data for
27; Figures S1−S3 (PDF)
0 mM ammonium acetate. The HPLC buffer B was 100% ACN.
The gradient was 80% B (0−1 min), 80−10% B (1−3 min), 10% B
(3−5 min), 10−80% B (5−5.3 min), and 80% B (5.3−10 min). The
column temperature was 40 °C, and the flow rate 0.5 mL/min. The
sample injection volume was 2 μL. The detection conditions of the
mass spectrometer were as follows: capillary voltage, 4000 V;
nebulizer pressure, 35 psi; cell accelerate voltage, 4 V; sheath gas
temperature, 400 °C; sheath gas flow, 11 L/min; source gas
temperature, 300 °C; source gas flow, 11 L/min; fragmenter voltage,
AUTHOR INFORMATION
■
Corresponding Author
Michael J. Soth − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States; orcid.org/0000-
0002-2649-5537; Email: mjsoth@mdanderson.org
80 V; collision energy, 26 V (glutamate) and 14 V (glutamine).
Metabolites were detected by compound-specific multiple reaction
monitoring transition (MRM) and retention time (RT): Glutamine
Authors
1
3
(
4
m/z 147 > 84, RT 4.93 min), C5-Glutamine (m/z 152 > 88, RT
Kang Le − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
1
3
.93 min), Glutamate (m/z 148 > 84, RT 4.78 min), C5-Glutamate
(m/z 153 > 88, RT 4.78 min). The method was validated with an
analytical range of 10−5000 ng/mL for both glutamine and
glutamate in ACN/water (1:1). GraphPad Prism was used for
generation of graphs, and data are expressed as mean ± standard
deviation.
Maria Emilia Di Francesco − Institute for Applied Cancer
Science (IACS), The University of Texas MD Anderson Cancer
Center, Houston, Texas 77030, United States
Matthew M. Hamilton − Institute for Applied Cancer Science
Efficacy in Ru337 PDX Model. NSG female mice (8 weeks old,
Jackson Labs) were implanted with Ru337 (Memorial Sloan
Kettering Cancer Center) patient-derived xenografts (PDX)
subcutaneously on the right flank. Tumors were allowed to grow
(
IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Gang Liu − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Jason P. Burke − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Chris L. Carroll − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Jeffrey J. Kovacs − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
3
to an average volume of 100 mm , as monitored by caliper
measurements. The animals were then randomized into groups of
eight. All of the animals received chow ad libitum. The mice were
treated with compounds on a 5 day-on/2 day-off schedule.
Compound 27 was formulated in 0.5% methylcellulose in sterile
water and dosed at 100 mg/kg, PO, BID (doses administered
approximately 8 h apart (8:00 and 16:00) each dosing day followed
by a 16 h gap before the next day’s dose). TAK-228 was formulated
in 5% sucrose and 0.5% methylcellulose in sterile water and dosed at
1
mg/kg, PO, QD. Bodyweights were monitored twice per week.
2
Tumor volume was calculated using the formula: V = l × L/2 (l =
length; L = width). GraphPad Prism was used for generation of
graphs, and data are expressed as mean ± standard deviation. For the
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c01398
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Jennifer P. Bardenhagen − Institute for Applied Cancer
Science (IACS), The University of Texas MD Anderson Cancer
Center, Houston, Texas 77030, United States
Christopher A. Bristow − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Mario Cardozo − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Barbara Czako − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States; orcid.org/0000-
Zhijun Kang − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Paul G. Leonard − Institute for Applied Cancer Science
(IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Zhen Liu − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Timothy McAfoos − Institute for Applied Cancer Science
(IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Meredith Miller − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Pietro Morlacchi − Institute for Applied Cancer Science
(IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Robert A. Mullinax − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Wylie S. Palmer − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
0
002-3357-7514
Elisa de Stanchina − Antitumor Assessment Core Facility
Molecular Pharmacology Program, Memorial Sloan Kettering
Cancer Center, New York, New York 10065, United States
Ningping Feng − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Jill R. Garvey − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Jason P. Gay − Translational Research to Advance Therapeutics
and Innovation in Oncology (TRACTION), The University of
Texas MD Anderson Cancer Center, Houston, Texas 77030,
United States
Jihai Pang − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Mary K. Geck Do − Institute for Applied Cancer Science
Norma Rogers − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
(
IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Jennifer Greer − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Michelle Han − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Angela Harris − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Charles M. Rudin − Drunkenmiller Center for Lung Cancer
Research, Memorial Sloan Kettering Cancer Center, New York,
New York 10065, United States
Hannah E. Shepard − Institute for Applied Cancer Science
(IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Nakia D. Spencer − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Jay Theroff − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Zachary Herrera − Institute for Applied Cancer Science
(IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Qi Wu − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Alan Xu − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Ju Anne Yau − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Giulio Draetta − Institute for Applied Cancer Science (IACS)
and Translational Research to Advance Therapeutics and
Innovation in Oncology (TRACTION), The University of
Texas MD Anderson Cancer Center, Houston, Texas 77030,
United States
Sha Huang − Institute for Applied Cancer Science (IACS), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Virginia Giuliani − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Yongying Jiang − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
Sarah B. Johnson − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Carlo Toniatti − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Troy A. Johnson − Institute for Applied Cancer Science
(
IACS), The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01398
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Timothy P. Heffernan − Translational Research to Advance
Therapeutics and Innovation in Oncology (TRACTION), The
University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, United States
Philip Jones − Institute for Applied Cancer Science (IACS),
The University of Texas MD Anderson Cancer Center,
Houston, Texas 77030, United States
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Notes
The authors declare the following competing financial
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ACKNOWLEDGMENTS
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Sun, I.-H.; Arwood, M. L.; Bettencourt, I. A.; Patel, C. H.; Wen, J.;
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chemistry department for helpful discussions throughout the
project. They also thank Jason Cross in their structural
chemistry department for preparing the modeling snapshot.
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San
́
■
Prigge, J. R.; Schmidt, E. E.; Thomas, C. J.; Goparaju, C.; Davies, A.;
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ACN, acetonitrile; AcOH, acetic acid; AUC, area under the
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1
,2,4-thiadiazol-2-yl)ethyl sulfide; Cl/CL, clearance; Cmax
maximum concentration; CYP, cytochrome P450; DAST,
diethylamino)sulfur trifluoride; DCM, dichloromethane;
DIPEA, N,N-diisopropylethylamine; DMAP, 4-dimethylami-
nopyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; DON, 6-diazo-5-oxy-L-norleucine; EDC, 1-ethyl-3-
,
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(
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(
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